这项研究是支持公共卫生服务补助金的AI - 34431（DAH）由美国国立过敏和传染病研究所和VA医学研究基金（DAH）。

1。 钩端螺旋体固定玻片<ol><li>钩端螺旋体是一种类BSL2病原体。活细胞的工作，它需要适当的处理，如戴手套，实验室外套，并在无菌罩移液步骤。 </li><li>在EMJH medium11成长的钩端螺旋体，辅以1％兔血清在30 ° C，直到他们达到月中下旬数期约6天（5 × 10 8细胞/ ml密度为5 × 10 7） 。 </li><li> 〜2000年7分钟在室温XG离心收获的文化。 </li><li>移除吸上清，轻轻重悬沉淀磷酸盐缓冲液（PBS）-5毫米氯化镁2，PH7.2到终浓度为5 × 10 8个细胞/ ml 。 </li><li>加入1 ml的细胞悬液，每孔两腔玻片和孵化在30 ° C至80分钟让细胞坚持。 </li><li>小心地取出液体含有未结合的细胞吸。 </li><li>修复剩余的细菌玻片，加入1 / 2％多聚甲醛PBS - 5毫米氯化镁2毫升。孵育40分钟，在30 ° C.这些幻灯片将用于评估暴露于表面的蛋白质。 </li><li>评估子表面蛋白对于控制幻灯片，通透冰冷的甲醇1毫升/ 100％固定的外膜。在-20 ° C孵育20分钟。甲醇行为在几个方面:permeabilizes外膜，denaturates的蛋白质，并修复细胞玻片。 </li><li>取出固定吸剂。 </li></ol> 2。标记的特异性抗体<ol><li>阻止非特异性结合，通过加入1毫升/封闭液（DIFCO钩端螺旋体浓缩EMJH）。在30 ° C孵育90分钟。 </li><li>稀释的特异性抗体（免疫兔血清或小鼠单克隆抗体，这里多克隆兔血清具体OmpL54 10，FlaA1 12，和OmpL1 13）和免疫前兔血清或鼠标腹水抗体（作为阴性对照时使用）不包含在封闭液。每个抗体的稀释液有经验取决于抗体滴度，抗原抗体反应，在细胞中的蛋白丰度加以确定。通常的范围是1:50到1:600​​。 </li><li>删除愿望封闭液，并添加1毫升/孔的稀释的抗体。 </li><li>在30 ° C孵育1小时。 </li><li>移除吸液和洗井三次，用PBS（1毫升/孔）。 </li></ol> 3。可视化的leptospires <ol><li>添加1毫升/清楚的Alexa Fluor 488标记（任羊抗兔IgG或羊抗鼠IgG）抗体稀释1:2000和荧光核酸染色，4&#39;6 - diamidino - 2 -苯基吲哚盐酸盐（ DAPI）的稀释到终浓度为0.25微克/毫升封闭液。此步骤可确保检测抗体结合的螺旋体抗体独立的约束力，分别的存在。 </li><li>孵育30℃，45分钟的幻灯片。 </li><li>移除吸出的液体和两次用PBS，一旦洗井，用蒸馏水（1毫升/孔）。 </li><li>删除商会及胶条从载玻片和空气干燥〜10分钟。 </li><li>延长黄金防褪色的安装介质（2 × 20μL每张幻灯片）和一个24 × 50毫米盖玻片。 </li><li>在室温下在黑暗中孵育过夜。这一步是必要的，以便安装介质固化（硬化）。 </li><li>用指甲油密封盖玻片。 </li><li>可视化染色荧光显微镜使用青色/​​蓝色为DAPI染色检测过滤器和一个绿色的检测过滤器的Alexa Fluor 488。为了确保准确的样本的代表性是，确保您评估每个样本的整个腔面积。 </li><li>记录每个样品的代表性成像领域的数据。当成像的Alexa Fluor 488荧光，使用的所有样品相同的曝光时间。使用一致的曝光时间，以便更准确的比较与测试抗原与控制的结果。 </li></ol> 4。代表性的成果: 钩端螺旋体使用对表面和亚表面蛋白的抗体细胞表面的IFA结果的例子如图2所示。一个测试被认为是积极的，当大量的荧光是一个感兴趣的蛋白质表面暴露（图2A）在具有完整leptospires的样品检测。通常情况下，在积极的测试（在这里，表面暴露OmpL54），大致相同的荧光强度观察完好和通透的细胞（图2A），它必须包括针对地下使用的抗体阴性对照为外膜的完整性，最好是周质蛋白（如钩端螺旋体FlaA1，图2B）。只有当数据表明，外膜保持完好杜里吴实验，即对一个子表面蛋白的抗体还没有反应过来（无荧光或荧光弱得多透样品图。2B）完整的细胞，它可以得出结论，感兴趣的蛋白质（S）表面暴露。列入作为一个额外的负面控制与免疫前血清（多克隆抗体的情况下）的实验是必要的，明确的数据，应该不会产生大量荧光完好或通透的细胞（图2A，2C）。 DAPI染色是必要的可视化时，结果为阴性（没有的Alexa Fluor 488荧光）观察leptospires。碘化丙啶Counterstaining到DAPI是一个可以接受的替代品。 这种方法是定性的，而不是明亮的荧光定量，可能是由于多个表面，暴露在一个相对平等的丰盈的其他抗原的抗原，但较少的表面暴露的抗原表位识别的抗原表位。因此只能用荧光强度比较相同的抗体，如完好与透细胞和不同的蛋白质之间没有荧光强度，反应。这个区别是重要的，当一个含糊的结果是取得显着减少的荧光比透性细胞（图2C）在完整的细胞中观察到。这样的结果表明，蛋白质在分型面的位置（完整细胞与一些非特异性背景染色），抗体优先是具有约束力的非本地的暴露后甲醇通透或蛋白质的抗原表位，可能有多个本地化网站所描述的ERP和ELP蛋白质的另一种螺旋体，莱姆病螺旋 体 14 。在这两种情况下，像这样一个含糊的IFA结果要求，如表面生物素或表面蛋白水解的替代方法，以确定是否感兴趣的蛋白质表面暴露或不10。 <img src="/files/ftp_upload/2805/2805fig1.jpg" alt="图1"/> 图1。表面钩端螺旋体螺旋体免疫荧光检测的流程图。首先，1 ml的细胞悬液加入到每孔以及两玻片（每次实验至少​​有两个幻灯片），孵育leptospires坚持。下一步，leptospires固定在玻片完整的外膜（OM）和冷甲醇1毫升/ 100％的样品与通透OM样品中加入1 / 2％多聚甲醛（PFA）毫升。接下来，leptospires孵育认识到，随着地下蛋白（小学AB公司）在30 ° C为90分钟的对照抗体表面暴露蛋白的抗体。然后，初级抗体检测1毫升/的Alexa Fluor 488标记的二抗孵育。最后，荧光显微镜观察细胞。 <img src="/files/ftp_upload/2805/2805fig2.jpg" alt="图2"/> 图2表面免疫分析钩端螺旋体蛋白质。钩端螺旋体螺旋体免疫和免疫前的血清，探讨，并用DAPI染液配对阴性结果证明螺旋体的存在。绿色荧光过滤器是用来检测的Alexa Fluor 488标记的二级抗体结合表面暴露的蛋白质和蓝色/青色荧光过滤器检测的DAPI结合到细胞的DNA。一）Leptospires探索与认识的一个暴露表面的蛋白质，OmpL54 10抗体。二）细胞探测与打击地下一个周质蛋白，FlaA1（阴性对照）的抗体。三）探讨，OmpL1钩端螺旋体孔蛋白抗体的细胞。绑定leptospires兔血清检测的Alexa Fluor 488标记的羊抗兔IgG片段。所有图像都采取后4秒的长时间曝光。

<ol>
	<li>Achouak, W., Heulin, T., Pages, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+facets+of+bacterial+porins.">Multiple facets of bacterial porins.</a> FEMS Microbiol Lett. 199, 1-7 (2001).</li><li>Haake, D. A., Matsunaga, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+leptospiral+outer+membrane+and+description+of+three+novel+leptospiral+membrane+proteins.">Characterization of the leptospiral outer membrane and description of three novel leptospiral membrane proteins.</a> Infect Immun. 70, 4936-4945 (2002).</li><li>Haake, D. 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我们的表面IFA技术是用来展示各种borrelial 15，16和钩端螺旋体12，17种蛋白质的表面接触的相似。这里描述的，旨在最大限度地减少操纵细胞在努力保持外膜的完整性，同时利用优势钩端螺旋体细胞的倾向，坚持以玻片表面IFA方法的细节。该方法涉及的多聚甲醛浓度较低（2％和4％），外层膜稳定缓冲液（PBS 5毫米氯化镁 2）和更少的洗涤步骤洗衣机比以前公布的协议12，...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>双室玻片</td><td>实验室康</td><td> 177380 </td><td></td></tr><tr><td>兔血清</td><td>罗克兰Immunochemicals </td><td> D209 - 00 - 0100 </td><td></td></tr><tr><td> 钩端螺旋体浓缩EMJH </td><td> BD DIFCO </td><td> 279510 </td><td></td></tr><tr><td>的Alexa Fluor 488山羊抗兔IgG（H + L） </td><td> Invitrogen公司/分子探针</td><td> A - 11034 </td><td></td></tr><tr><td>的Alexa Fluor 488山羊抗小鼠IgG（H + L） </td><td> Invitrogen公司/分子探针</td><td> A - 11029 </td><td></td></tr><tr><td> 4&#39;6 - diamidino - 2 -苯基吲哚盐酸盐（DAPI） </td><td> Invitrogen公司/分子探针</td><td> D1306 </td><td></td></tr><tr><td>延长黄金防褪色的安装介质</td><td> Invitrogen公司/分子探针</td><td> P36930 </td><td>在使用前平衡至室温</td></tr><tr><td> Fisherfinest高级玻璃盖</td><td>费舍尔</td><td> 12-544-14 </td><td></td></tr><tr><td>荧光显微镜</td><td>蔡司</td><td> Axioskop 40 </td><td></td></tr></tbody></table>

immuno-fluorescence assay of leptospiral surface-exposed proteins

细菌表面蛋白与宿主细胞的直接接触和吸收营养物质从环境1的参与。出于这个原因，细胞定位，可以提供细菌蛋白质的功能作用的见解。表面细菌蛋白质的本地化是对识别致病的机制，涉及的致病因素关键步骤。 分馏2-5钩端螺旋体膜的方法可为一定阶级的外膜蛋白（OMPS），如脂蛋白与跨膜外膜蛋白，有选择性的，因此导致的错误分类。这可能是由于结构上的差异，以及它们是如何相关的外膜。脂蛋白与膜通过脂基团之间的N -末端（3脂肪酸）和脂质双层磷脂6，7的疏水相互作用。相比之下，跨膜外膜蛋白通常集成在一个筒状结构，8，9排列的双亲性β-张成脂质双层。此外，在外膜蛋白的存在并不一定能保证，暴露在表面上的蛋白质或它的域。 Spirochetal外膜被称为脆弱，因此必要的方法，涉及温柔操纵子表面蛋白细胞和列入控制评估外膜的完整性。 在这里，我们目前的免疫荧光法（IFA）的方法来直接评估完好leptospires的蛋白质表面暴露。这种方法是基于对钩体表面蛋白抗原特异性抗体识别。在这里，特定抗体OmpL54 10 detetcted aftero约束力的本地人，表面抗原决定簇暴露。抗体反应的比较完整的与透细胞，使细胞分布进行评估，并与否的一种蛋白质是钩体表面选择性。应分摊的外膜的完整性，使用一个或多个地下蛋白质，最好在位于周质抗体。 表面IFA方法可用于分析任何钩端螺旋体特异性抗体的蛋白质表面暴露。的实用性和方法的限制，取决于是否能够绑定到本地的抗原表位的抗体。由于抗体往往提出对重组蛋白抗原表位，原生的，暴露于表面的蛋白质可能无法识别。然而，表面的IFA方法是一个完整的细菌表面成分研究的宝贵工具。这种方法不仅可应用于leptospires而且其他螺旋体和革兰氏阴性菌。更强的外膜蛋白的表面接触的结论，是一个全面的方法，涉及多种细胞定位方法推荐10。

Watch this Scientific Journal Video about Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins at JoVE.com

Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

一种有效的方法来评估钩端螺旋体蛋白质表面暴露描述。该方法是专门设计，以避免破坏脆弱的钩体细胞的外膜。这种技术需要雇用几个阴性对照，以评估外膜的完整性和特异性抗体反应。

钩端螺旋体暴露于表面的蛋白免​​疫荧光法

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment 1. For this reason, cellular ...

immuno-fluorescence-assay-of-leptospiral-surface-exposed-proteins

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Immunology and Infection

15.7K 次观看.  University of California, Los Angeles. 一种有效的方法来评估钩端螺旋体蛋白质表面暴露描述。该方法是专门设计，以避免破坏脆弱的钩体细胞的外膜。这种技术需要雇用几个阴性对照，以评估外膜的完整性和特异性抗体反应。

视频: Immuno-fluorescence Assay of Leptospiral Surface-exposed Proteins

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

酶标菜生物膜形成实验

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

科研

免疫与感染

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

对小鼠诺沃克类病毒的空斑法

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

有效生成荧光标记疫苗病毒蛋白的方法

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

利用荧光蛋白监视非洲锥虫Glycosome动态

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学:生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.

The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.

高灵敏检测方法的沙粒，细胞附着的测量

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral ...

Optimization of a Quantitative Micro-neutralization Assay

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. In this study, we present the protocol of an imaging-based MN assay to quantify the true antigenic relationships between viruses. Unlike typical plaque reduction assays that rely on visible plaques, this assay quantitates the entire infected cell population of each well. The protocol matches the virus type or subtype with the selection of cell lines to achieve maximum infectivity, which enhances sample contrast during imaging and image processing. The introduction of quantitative titration defines the amount of input viruses of neutralization and enables the results from different experiments to be comparable. The imaging setup with a flatbed scanner and free downloadable software makes the approach high throughput, cost effective, user friendly, and easy to deploy in most laboratories. Our study demonstrates that the improved MN assay works well with the current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses, without being significantly influenced by amino acid substitutions in the neuraminidase (NA) of A(H3N2) viruses. It is particularly useful for the characterization of viruses that either grow to low HA titer and/or undergo an abortive infection resulting in an inability to form plaques in cultured cells.

This study describes an imaging-based micro-neutralization assay to analyze the antigenic relationships between viruses. The protocol employs a flatbed scanner and has four steps, including titration, titration quantitation, neutralization, and neutralization quantitation. The assay works well with current circulating influenza A(H1N1)pdm09, A(H3N2), and B viruses.

定量微中和试验的优化

The micro-neutralization (MN) assay is a standard technique for measuring the infectivity of the influenza virus and the inhibition of virus replication. ...

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. Whereas Pd transport of small molecules, such as ions and water, is presumed to occur passively, cell-to-cell transport of biological macromolecules, such proteins, most likely occurs via an active mechanism that involves specific targeting signals on the transported molecule. The scarcity of identified plasmodesmata (Pd) localization signals (PLSs) has severely restricted the understanding of protein-sorting pathways involved in plant cell-to-cell macromolecular transport and communication. From a wealth of plant endogenous and viral proteins known to traffic through Pd, only three PLSs have been reported to date, all of them from endogenous plant proteins. Thus, it is important to develop a reliable and systematic experimental strategy to identify a functional PLS sequence, that is both necessary and sufficient for Pd targeting, directly in the living plant cells. Here, we describe one such strategy using as a paradigm the cell-to-cell movement protein (MP) of the Tobacco mosaic virus (TMV). These experiments, that identified and characterized the first plant viral PLS, can be adapted for discovery of PLS sequences in most Pd-targeted proteins.

Identification of Plasmodesmal Localization Sequences in Proteins In Planta

Plant intercellular connections, the plasmodesmata (Pd), play central roles in plant physiology and plant-virus interactions. Critical to Pd transport are sorting signals that direct proteins to Pd. However, our knowledge about these sequences is still in its infancy. We describe a strategy to identify Pd localization signals in Pd-targeted proteins.

蛋白质中 Plasmodesmal 定位序列的识别在底中

Plasmodesmata (Pd) are cell-to-cell connections that function as gateways through which small and large molecules are transported between plant cells. ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and cellular proteins by mass spectrometry. Although proteins that interact with viral genomes play major roles in determining the outcome of infection, a comprehensive analysis of viral genome associated proteins was not previously feasible. Here we demonstrate a method that enables the direct purification of HSV-1 genomes from infected cells. Replicating viral DNA is selectively labeled with modified nucleotides that contain an alkyne functional group. Labeled DNA is then specifically and irreversibly tagged via the covalent attachment of biotin azide via a copper(I)-catalyzed azide-alkyne cycloaddition or click reaction. Biotin-tagged DNA is purified on streptavidin-coated beads and associated proteins are eluted and identified by mass spectrometry. This method enables the selective targeting and isolation of HSV-1 replication forks or whole genomes from complex biological environments. Furthermore, adaptation of this approach will allow for the investigation of various aspects of herpesviral infection, as well as the examination of the genomes of other DNA viruses.

The goal of this protocol is to specifically tag and selectively isolate viral DNA from infected cells for the characterization of viral genome associated proteins.

钩端螺旋体暴露于表面的蛋白免​​疫荧光法

钩端螺旋体暴露于表面的蛋白免疫荧光法